The human Peripheral Benzodiazepine Receptor (hPBR) is a highly hydrophobic, tryptophan-rich (7.7%) 18kDa protein consisting of 169 amino acids and five predicted transmembrane a-helices. hPBR is primarily localized within the mitochondrial outer membrane, where it is one component of the mitochondrial permeability transition pore complex. Although the primary cellular function of hPBR within the cell is not currently known, it has been implicated in a variety of cellular functions such as cellular proliferation and apoptosis, making hPBR a target for cancer therapy, hPBR has been cloned into an E. coli expression system, expression has been optimized, and reproducible high-level expression has been achieved. A robust two column purification method has been developed that enables the facile generation of multimilligram quantities of highly pure hPBR. The aims of this R21 application, submitted under Program Announcement PA-03-100, NIGMS Exploratory Studies for High Impact/High Risk Research, are to: 1) produce multi-milligram quantities of purified hPBR, and 2) conduct systematic three-dimensional crystallization experiments with hPBR and hPBR complexes. The goal of this proposal is to obtain crystals sufficient for structure determination of hPBR by x-ray crystallography. The longer-term goals of this effort are to conduct detailed structure-function studies of hPBR, to critically evaluate the utility of the hPBR structure for design of novel cancer drugs, and to extend our research to the structural biology of the mitochondrial permeability transition pore complex.